1. Field of the Invention
The present invention relates to a midecamycin hyper producing strain having improved productivity of midecamycin which is a member of macrolide antibiotics, and a method for producing midecamycins using this strain.
2. Background Art
Macrolide antibiotics are antibacterial agents effective upon Gram-positive bacteria, Mycoplasma, Chlamydia and the like and classified into clinically important antibacterial agents because they can be orally administered and have low toxicity. Among them, commercially available 16-membered ring macrolide antibiotics are broadly used in the world including Asian countries, because they have advantages in that resistances are hardly induced, interaction with other drugs is less in comparison with 14-membered ring macrolide antibiotics and influence upon the intestinal tract is also less.
Midecamycins (FIG. 1) are 16-membered ring macrolide antibiotics produced by Streptomyces mycarofaciens (ATCC 21454) and the like species of actinomysetes. Miocamycin as an acylation derivative thereof (Omoto, S. et al., J. Antibiot., 29, 536 (1976); Yoshida, T. et al., Jpn. J. Antibiot., 35, 1462 (1982)) is clinically broadly used and produced from a fermentation product of Streptomyces mycarofaciens. In addition, since Streptomyces mycarofaciens does not produce leucomycins, it also has an advantage in that removal of leucomycins by purification and the like steps can be omitted by the use of this strain.
Actinomysetes have been occupying an important position for a long time in the field of fermentation industries as producer strains of antibiotics, physiologically active substances and the like secondary metabolites, and improvement of their productivities have been carried out by various strain breeding techniques. Strain breeding by mutagenesis using various mutagens has also been carried out on the production of midecamycin by Streptomyces mycarofaciens. According to such a strain breeding method, it has an advantage in that a strain having a phenotype of interest can be conveniently obtained, but it cannot be elucidated about what a type of mutation was introduced into which gene. As a result of introducing random mutation, there is a possibility that a mutation that is not useful (e.g., not increasing a productivity) is jointly introduced in the breeding thereafter.
From such a point of view, it is possible to extract a useful mutation by comparing genomic sequence of a low production strain with that of a hyper producing strain, and it is possible to create a hyper producing strain in which a useful mutation alone is accumulated making use of recombinant DNA techniques.
In the microorganisms which produce macrolide antibiotics, the majority of macrolide biosynthesis genes are together concentrated within a region of from 70 to 80 kb of the genome in many cases (Donadio, S. et al., Science, 252, 675 (1991); MacNeil, D. J. et al., Gene, 115, 119 (1992); Schwecke, T. et al., Proc. Natl. Acad. Sci., 92, 7839 (1995)). A high homology gene coding for a giant multifunctional protein called type I polyketide synthase (to be referred also to as PKS hereinafter) is present in the center of their cluster.
The PKS gene is generally constituted from 3 to 5 genes, and its protein forms a complex consisting of an initiator module and several extender modules. Each of them adds a specific acyl-CoA precursor to a polyketide chain in the middle of its synthesis and specifically modifies β-keto group. Accordingly, the polyketide structure is determined by the composition and order of these modules in PKS. The modules contain several domains, and each of them carries out specified function.
The initiator module consists of an acyl carrier protein (to be referred to as ACP hereinafter) domain to which acyl group of the precursor binds and an acyl transferase (to be referred to as AT hereinafter) domain which catalyzes addition of acyl group to the ACP domain. Depending on the specificity of this AT domain, the kind of acyl-CoA to be added thereto is determined. All of the extender modules contain a β-ketosynthase (β-ketoacyl acyl carrier protein synthase, to be referred also to as KS hereinafter) domain which adds the previously presenting polyketide chain to new acyl-ACP by decarboxylation condensation, and AT domain and ACP domain.
Also, each of the extender modules contain some of the domains which modify specific β-keto groups, in addition to these, and modification of the β-keto groups is determined based on the structure of the domain to be contained. These include a β-ketoreductase (to be referred also to as KR hereinafter) domain which reduces β-keto group to hydroxyl group, a dehydratase (to be referred also to as DH hereinafter) domain that removes hydroxyl group and forms double bond and an enoyl reductase (to be referred also to as ER hereinafter) domain which forms saturated carbon bond.
The last extender module is completed with a thioesterase (to be referred also to as TE hereinafter) domain which releases polyketide through its cyclization from PKS. Boundaries of modules, domains and open reading frame (to be referred also to as ORF hereinafter) of PKS can be estimated based on the sequence information on already known PKS genes.
The polyketide backbone formed by PKS undergoes methylation, acylation, oxidation, reduction, specific sugar addition and the like additional modifications, and a macrolide antibiotic is finally synthesized. Most of the genes necessary for these modifications are present in the periphery of the PKS gene.
Genes which encode deoxy sugar biosynthesis enzymes have been revealed regarding erythromycin, tylosin and the like (Summers, R. G. et al., Microbiology, 143, 3251 (1997);Gaisser, S. et al., Mol. Gen. Genet., 256, 239 (1997); Merson-Davies, L. A. and Cundliffe, E., Mol. Microbiol., 13, 349 (1994)). Synthesis of these deoxy sugars comprises activation of glucose by the addition of nucleotide diphosphate and subsequent deoxygenation, reduction, epimerization, amination, methylation and the like reactions. These sugars are introduced into macrolides by the action of specific glycosyltransferase.
Since the structures of midecamycin bear resemblance to the structures of tylosin, it is considered that it passes through almost the same biosynthetic pathway. Biosynthesis of midecamycin starts with the synthesis of malonyl-CoA, methyl malonyl-CoA, ethyl malonyl-CoA and methoxy malonyl-CoA which are precursors of the polyketide backbone. These precursors undergo cyclization through the stepwise condensation reaction by polyketide synthase, and the polyketide backbone is synthesized as a result. Thereafter, midecamycin is finally synthesized via sugar saccharide addition, hydroxylation, formylation, acylation and the like modification reactions.
In order to improve its productivity by gene recombination techniques, expression reinforcement of genes encoding the rate-determining biosynthesis reactions, expression reinforcement or gene disruption of genes which regulate expression of the biosynthesis genes, interception of unnecessary secondary metabolism systems and the like have been carried out (Kennedy, J. and Turner, G., Mol. Gen. Genet., 253, 189 (1996); Review: Balts, R. H., Biotechnology of Antibiotics Second Edition, Revised and Expanded, Marcel Dekker, Inc., New York, p. 49 (1997); Review: Hutchinson, C. R. and Colombo, A. L., Ind. Microbiol. Biotechnol., 23, 647 (1999); Review: Brakhage, A. A., Microbiol. Mol. Biol. Rev., 62, 547 (1998)). Accordingly, when the biosynthesis gene is specified, the productivity can be improved by means of gene recombination techniques, by connecting it to an appropriate vector and introducing into a secondary metabolite producing strain.